Treatment of degenerated disc with autologous cells

ABSTRACT

The present invention relates to administering autologous uncultured cells into a diseased intervertebral disc.

RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.10/714,594, filed Nov. 14, 2003, which is a continuation-in-partapplication of U.S. application Ser. No. 10/714,559, filed Nov. 13,2003, now abandoned, which is a continuation-in-part of U.S. applicationSer. No. 10/631,487, filed Jul. 31, 2003, which is acontinuation-in-part application of U.S. application Ser. No.10/610,355, filed Jun. 30, 2003 now U.S. Pat. No. 7,429,378, which is acontinuation-in-part application of U.S. application Ser. No.10/456,948, filed Jun. 6, 2003 now U.S. Pat. No. 7,344,716, which claimsthe benefit of priority from U.S. Provisional Application No.60/470,098, filed May 13, 2003. The entire teachings of the aboveapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The natural intervertebral disc contains a jelly-like nucleus pulposussurrounded by a fibrous annulus fibrosus. Under an axial load, thenucleus pulposus compresses and radially transfers that load to theannulus fibrosus. The laminated nature of the annulus fibrosus providesit with a high tensile strength and so allows it to expand radially inresponse to this transferred load.

In a healthy intervertebral disc, the cells within the nucleus pulposusform only about one percent of the disc tissue by volume. These cellsproduce an extracellular matrix (ECM) containing a high percentage ofproteoglycans. These proteoglycans contain sulfated functional groupsthat retain water, thereby providing the nucleus pulposus with itscushioning qualities. The nucleus pulposus cells may also secrete smallamounts of cytokines as well as matrix metalloproteinases (MMPs). Thesecytokines and MMPs help regulate the metabolism of the nucleus pulposuscells.

In some instances of disc degeneration disease (DDD), gradualdegeneration of the intervertebral disc is caused by mechanicalinstabilities in other portions of the spine. In these instances,increased loads and pressures on the nucleus pulposus cause the cellswithin the disc (or invading macrophages) to emit larger than normalamounts of the above-mentioned cytokines. In other instances of DDD,genetic factors or apoptosis can also cause a decline in the number ofdisc cells and/or release of toxic amounts of these cytokines and MMPs.In some instances, the pumping action of the disc may malfunction (dueto, for example, a decrease in the proteoglycan concentration within thenucleus pulposus), thereby retarding the flow of nutrients into the discas well as the flow of waste products out of the disc. This reducedcapacity to provide nutrients to the cells and eliminate waste mayresult in decreased cell viability and metabolism resulting in furtherdegradation of the ECM along with the accumulation of high levels oftoxins that may cause nerve irritation and pain.

As DDD progresses, toxic levels of the cytokines and MMPs present in thenucleus pulposus begin to degrade the ECM. In particular, the MMPs (asmediated by the cytokines) begin cleaving the water-retaining portionsof the proteoglycans, thereby reducing its water-retaining capabilities.This degradation leads to a less flexible nucleus pulposus, and sochanges the loading pattern within the disc, thereby possibly causingdelamination of the annulus fibrosus. These changes cause moremechanical instability, thereby causing the cells to emit even morecytokines, typically thereby upregulating MMPs. As this destructivecascade continues and DDD further progresses, the disc begins to bulge(“a herniated disc”), and then ultimately ruptures, causing the nucleuspulposus to contact the spinal cord and produce pain.

U.S. Pat. No. 6,352,557 (“Ferree”) teaches adding therapeutic substancessuch as nucleus pulposus cells to morselized extra-cellular matrixobtained from donors, and injecting that combination into anintervertebral disc. However, the cells first need to be cultured andthen added to the donor matrix prior to implantation into the diseaseddisc. This process requires a delay in the patient's treatment inaddition to subjecting the patient to two separate procedures. The firstprocedure is to harvest the cells, which then require culturing.Following the culturing the cells are implanted into the patient.

U.S. Pat. No. 6,340,369 (“Ferree II”) teaches harvesting liveintervertebral disc cells from a patient, culturing the cells andtransplanting them into the affected disc. Ferree II further teachesthat the cells can be combined with Type II collagen-glycosaminoglycanmatrix or Type I collagen-glycosaminoglycan matrix depending on whetherthe cells are harvested from the nucleus pulposus (NP) or annulusfibrosus (AF). Also Ferree II suggests adding one or more therapeuticsubstances to cells prior to transplantation. As an alternate source forcells, Ferree proposes using precursor cells of NP or AF cells,chondrocytes or other living cells that function like or coulddifferentiate into NP or AF cells. Throughout, Ferree teaches that theharvested cells are cultured prior to transplantation.

Alini, Eur. Spine J., 11 (Supp. 2): S215-220 (2002), suggests thatinjection of a biomatrix embedded with cells will have the potential torestore functionality to the disc. Alini's experiments are directed toisolating cells from the nucleus pulposus and culturing them. Alini alsosuggests other sources of cells including disc cells from allogenicdonors and autologous stem cells. His teachings suggest that stem cellswould be an ideal source but that there are no known methods forculturing the stem cells such that they would differentiate into nucleuspulposus cells prior to implantation. In essence, Alini requires thatcells be cultured prior to implantation.

Russell (Abstract 27 ISSLS 2003) reports conducting an experiment todetermine whether mesenchymal stem cells (MSCs) could be directed topresent disc chondrocyte phenotypes. Russell found that adult human MSCswere induced to differentiate along a chondrocytic phenotype whenmediated by culture conditions and also by addition of TGF-B1.

Sakai (Abstract 24 ISSLS 2003) reports evaluating whether autologoustransplantation of MSCs to the disc would prevent disc degeneration.Using rabbits, MSCs were isolated from the bone marrow and cultured for2 weeks prior to transplantation. Results showed significant discpreservation.

Sakai, Biomaterials, 24: 3531-3541 (2003) describes using a final celldensity of 1×10⁶ cells/ml, to inject 0.04 ml of solution in whichautogenous cultured MSCs were embedded through a 27-gauge insulininjector to each disc. Proliferation of cells after transplantation wasfound to be successful.

Sobajima (Abstract 43 ISSLS 2002) studied the feasibility of stem celltherapy for DDD. Human NP cells were isolated from patients undergoingdisc surgery and were co-cultured with either MSCs from patientsundergoing hip surgery or muscle derived stem cells from mice. The datademonstrated a synergistic effect between stem cells and nucleuspulposus cells, resulting in upregulated proteoglycan synthesis in vivo.

Ganey, Eur Spine J, 11 (Suppl. 2):S206-S214 (2002), reported onsurgeries conducted in Germany where cells were harvested from portionsof a patient's disc after discectomy. The cells were then cultured andreturned for transplantation into the patient at a later date.

Sander et al. in US Patent Application Publication 2003/0069639, teachesusing tissue biopsies taken from a patient as a source to harvest cellsfor implantation into a degenerated disc.

All of the teachings cited above require culturing of cells prior toimplantation, which, in turn, necessitates a delay in treating thepatient's degenerating disc.

SUMMARY OF THE INVENTION

The present inventors have developed an intra-operative procedure forefficaciously treating degenerative disc disease by introducingautologous uncultured cells, (e.g., mesenchymal stem cells orchondrocytes or fibroblasts) into the patient's disc. This procedureprovides immediate point of care treatment for the patient.

In accordance with one embodiment of the present invention, the presentinventors have developed a method of treating an intervertebral disc inwhich cells harvested from the patient's bone marrow are then introducedinto the degenerated disc to differentiate into nucleus pulposus and/orannulus fibrosus cells present in the disc, thereby increasing thenumber of those cells present in the disc. In some embodiments, theimplantation of the cells into the disc can occur immediately followingthe harvesting of the cells, so that the patient can avoid undergoing afirst procedure to harvest the cells, waiting for the cells to becultured (which may take several weeks), and then returning for a secondprocedure to implant the cultured cells into the disc.

There are believed to be several advantages to introducing cells to atargeted disc. A primary function of the cells is to produceextra-cellular matrix. As described above, there are several factorsthat result in cell death or malfunction, which in turn contribute tothe degradation of this matrix. One strategy to rebuild or regeneratethe extra-cellular matrix is to increase the number of viablefunctioning disc cells producing the matrix. The inventors believe thatthe plasticity phenomenon of the mesenchymal stem cells (MSCs) makesthem an ideal choice of cell type for differentiating into disc cellsafter implantation into the targeted disc. The cells may become nucleuspulposus (NP) and/or annulus fibrosus (AF) cells that will be capable ofproducing the necessary extra-cellular matrix within the disc. Inaddition, at the time of implantation, the cells may be combined withother therapeutic agents such as growth factors to help the cellssurvive, once inside the disc.

Accordingly, in one aspect of the present invention, there is provided amethod of treating degenerative disc disease in an intervertebral disc,comprising harvesting MSCs from a patient, and introducing the viableMSCs, without having to culture them, into the same patient'sdegenerated intervertebral disc, where the cells will proliferate anddifferentiate into nucleus pulposus and/or annulus fibrosus cells.

In some embodiments, the cells are delivered alone or via a carrier. Inother embodiments, the cells are delivered along with an additionaltherapeutic agent or substance such as a growth factor to the disc.

DETAILED DESCRIPTION OF THE INVENTION

Because DDD is a continuous process, the degenerating disc to which thecells are administered may be in any one of a number of degenerativestates. Accordingly, the degenerating disc may be an intact disc. Thedegenerating disc may be a herniated disc (i.e., wherein a portion ofthe annulus fibrosus has a bulge). The degenerating disc may be aruptured disc (i.e., wherein the annulus fibrosus has ruptured and thebulk nucleus pulposus has exuded). The degenerating disc may bedelaminated (i.e., wherein adjacent layers of the annulus fibrosus haveseparated). The degenerating disc may have fissures (i.e., wherein theannulus fibrosus has fine cracks or tears through which selectedmolecules from the nucleus pulposus can leak). In all of thesedegenerative states, the extra-cellular matrix of either the AF or NP isalso degrading.

The present invention is directed to intra-operatively providinghealthy, viable autologous mesenchymal stem cells (MSCs) to adegenerated intervertebral disc of a patient. The cells may be deliveredto either the nucleus pulposus or the annulus fibrosus or both forrepair and restoration of each respective extra-cellular matrix.

The inventors believe that MSCs provide a special advantage foradministration into a degenerating disc because they possess propertiesthat will help them to more readily survive the relatively harshenvironment present in the degenerating disc. Specifically, MSCs have adesirable level of plasticity that gives them the ability to proliferateand differentiate into NP and AF cells.

In one embodiment, the MSCs are obtained from the patient's own bonemarrow. In other embodiments, adipose or muscle tissue may be the sourceof MSCs. In some embodiments, the MSCs to be administered to the discare provided in a concentrated form. When provided in concentrated form,the cells can be uncultured. Uncultured, concentrated MSCs can bereadily obtained by centrifugation, filtration (selective retention), orimmunoabsorption. When filtration is selected, the methods disclosed inU.S. Pat. No. 6,049,026 (“Muschler”), the contents of which areincorporated by reference in their entirety, can be used. In someembodiments, the matrix used to filter and concentrate the MSCs is alsoco-administered into the nucleus pulposus or annulus fibrosus as atherapeutic agent. If this matrix has suitable mechanical properties, itcan be used to restore the height of the disc space that was lost duringthe degradation process. The cells may be injected at the same time orconcurrently with the matrix in the targeted area of the disc.

The volume of aspirated bone marrow obtained to harvest the MSCs ispreferably between about 5 cc to about 100 cc. This volume is then usedduring the concentration process to concentrate the MSCs.

When centrifugation is selected, the methods disclosed by Connolly etal. can be used. Incorporated by reference in its entirety isDevelopment of an Osteogenic Bone Marrow Preparation, JBJS 71-A (No. 5)(June 1989). In this rabbit study, Connolly reported that centrifugationof 7-10 ml of bone marrow yielded an average of 3.6×10⁶ nucleated cellsper milliliter in final cell suspension.

When the cells are concentrated using the centrifugation process, theyare deliverable to the disc in a pellet form in suspension. In anotherembodiment, the cells are delivered using a carrier. The carrier cancomprise, or can be selected from, the group consisting of beads,microspheres, nanospheres, hydrogels, gels, polymers, ceramics, collagenand platelet gels.

The carrier, in solid or fluid form, can carry the cells in severaldifferent ways. The cells can be embedded, encapsulated, suspended orattached to the surface of the carrier. In one embodiment, the carrierencapsulates the cells, provides nutrients, and protects the cells whenthey are delivered inside the disc. After a period of time inside thedisc, the carrier degrades and releases the cells. Specific types of thevarious carriers are described below.

In some embodiments, the mesenchymal stem cells are provided in asustained release device (i.e., sustained delivery device). Theadministered formulation can comprise the sustained release device. Thesustained release device is adapted to remain within the disc for aprolonged period and slowly release the mesenchymal stem cells containedtherein to the surrounding environment. This mode of delivery allows themesenchymal stem cells to remain in therapeutically effective amountswithin the disc for a prolonged period. One or more additionaltherapeutic agents can also be delivered by a sustained delivery device.

Synthetic scaffolds, such as fumaric-acid based scaffolds, have beendesigned and tailored to allow for attraction of certain cells and toprovide direction for the cells to differentiate in desired areas. Thecells can also be embedded in the scaffold and then injected into thetarget area without affecting the viability or proliferation of thecells. After implantation of the fumaric-acid based scaffold, itdegrades over time and no further surgery is necessary to remove thescaffold.

Carriers can also comprise hydrogels. The cells are encapsulated in thepolymer chains of the hydrogel after gelation. Hydrogels can bedelivered in a minimally invasive manner, such as injection to thetarget area. The hydrogel is also resorbed by the body. Hydrogelproperties such as degradation time, cell adhesion behavior and spatialaccumulation of extracellular matrix can be altered through chemical andprocessing modifications.

Hydrogels suitable for use in the present invention includewater-containing gels, i.e., polymers characterized by hydrophilicityand insolubility in water. See, for instance, “Hydrogels”, pages458-459, in Concise Encyclopedia of Polymer Science and Engineering,Eds. Mark et al., Wiley and Sons (1990), the disclosure of which isincorporated herein by reference in its entirety. Although their use isoptional in the present invention, the inclusion of hydrogels can behighly advantageous since they tend to possess a number of desirablequalities. By virtue of their hydrophilic, water-containing nature,hydrogels can house viable cells, such as mesenchymal stem cells, andcan assist with load bearing capabilities of the disc.

In one embodiment, the hydrogel is a fine, powdery synthetic hydrogel.Suitable hydrogels exhibit an optimal combination of properties such ascompatibility with the matrix polymer of choice, and biocompatability.The hydrogel can include any one or more of the following:polysaccharides, proteins, polyphosphazenes,poly(oxyethylene)-poly(oxypropylene) block polymers,poly(oxyethylene)-poly(oxypropylene) block polymers of ethylene diamine,poly(acrylic acids), poly(methacrylic acids), copolymers of acrylic acidand methacrylic acid, poly(vinyl acetate), and sulfonated polymers.

In general, these polymers are at least partially soluble in aqueoussolutions, e.g., water, or aqueous alcohol solutions that have chargedside groups, or a monovalent ionic salt thereof. There are many examplesof polymers with acidic side groups that can be reacted with cations,e.g., poly(phosphazenes), poly(acrylic acids), and poly(methacrylicacids). Examples of acidic groups include carboxylic acid groups,sulfonic acid groups, and halogenated (preferably fluorinated) alcoholgroups. Examples of polymers with basic side groups that can react withanions are poly(vinyl amines), poly(vinyl pyridine), and poly(vinylimidazole).

In accordance with the present invention, there is provided a method oftreating degenerative disc disease in an intervertebral disc having anucleus pulposus, comprising administering autologous unculturedmesenchymal stem cells into a degenerated intervertebral disc.

In one embodiment, the autologous mesenchymal stem cells are harvestedbefore they are administered into the disc.

In accordance with one aspect of the invention, the mesenchymal stemcells can be delivered into the disc space with at least one (an)additional therapeutic agent, such as an agent to aid in theproliferation and differentiation of the cells. There can be, forexample, one additional therapeutic agent (i.e., a second therapeuticagent) or there can be multiple additional therapeutic agents (e.g.,second and third therapeutic agents). The additional therapeutic agentmay be delivered simultaneously with the mesenchymal stem cells. Inanother embodiment, the additional therapeutic agent is delivered afteradministering the mesenchymal stem cells to the disc. In yet another,the additional therapeutic agent is administered first, i.e., prior toadministering the mesenchymal stem cells to the disc.

The same carrier may also be used to deliver the cells and theadditional therapeutic agent. In some embodiments, the cells are locatedon the surface of the carrier and the additional therapeutic agent isplaced inside the carrier. In other embodiments, the cells and theadditional therapeutic agent may be delivered using different carriers.

Other additional therapeutic agents which may be added to the discinclude, but are not limited to: vitamins and other nutritionalsupplements; hormones; glycoproteins; fibronectin; peptides andproteins; carbohydrates (simple and/or complex); proteoglycans;oligonucleotides (sense and/or antisense DNA and/or RNA); bonemorphogenetic proteins (BMPs); differentiation factors; antibodies (forexample, antibodies to infectious agents, tumors, drugs or hormones);gene therapy reagents; and anti-cancer agents. Genetically altered cellsand/or other cells may also be included in the matrix of this invention.If desired, substances such as pain killers (i.e., analgesics) andnarcotics may also be admixed with the carrier for delivery and releaseto the disc space.

In some embodiments, growth factors are additional therapeutic agents.As used herein, the term “growth factor” encompasses any cellularproduct that modulates the growth or differentiation of other cells,particularly connective tissue progenitor cells. The growth factors thatmay be used in accordance with the present invention include, but arenot limited to, members of the fibroblast growth factor family,including acidic and basic fibroblast growth factor (FGF-1 and FGF-2)and FGF-4, members of the platelet-derived growth factor (PDGF) family,including PDGF-AB, PDGF-BB and PDGF-AA; EGFs, members of theinsulin-like growth factor (IGF) family, including IGF-I and -II; theTGF-β superfamily, including TGF-β1, 2 and 3 (including MP-52),osteoid-inducing factor (OIF), angiogenin(s), endothelins, hepatocytegrowth factor and keratinocyte growth factor; members of the bonemorphogenetic proteins (BMPs) BMP-1, BMP-3, BMP-2, OP-1, BMP-2A, BMP-2B,BMP-4, BMP-7 and BMP-14; HBGF-1 and HBGF-2; growth differentiationfactors (GDFs), members of the hedgehog family of proteins, includingindian, sonic and desert hedgehog; ADMP-1; GDF-5; and members of thecolony-stimulating factor (CSF) family, including CSF-1, G-CSF, andGM-CSF; and isoforms thereof. The growth factor can be autologous suchas those included in platelet rich plasma or obtained commercially. Inone embodiment, the growth factor is administered in an amount effectiveto repair disc tissue.

In some embodiments, the growth factor is selected from the groupconsisting of TGF-β, bFGF, and IGF-1. These growth factors are believedto promote regeneration of the nucleus pulposus, or stimulateproliferation and/or differentiation of chondrocytes, as well asextracellular matrix secretion. In one embodiment, the growth factor isTGF-β. More preferably, TGF-β is administered in an amount of betweenabout 10 ng/ml and about 5000 ng/ml, for example, between about 50 ng/mland about 500 ng/ml, e.g., between about 100 ng/ml and about 300 ng/ml.

In one embodiment, at least one of the additional therapeutic agents isTGF-β1. In one embodiment, another additional therapeutic agent is FGF.

In some embodiments, platelet concentrate is provided as an additionaltherapeutic agent. In one embodiment, the growth factors released by theplatelets are present in an amount at least two-fold (e.g., four-fold)greater than the amount found in the blood from which the platelets weretaken. In some embodiments, the platelet concentrate is autologous. Insome embodiments, the platelet concentrate is platelet rich plasma(PRP). PRP is advantageous because it contains growth factors that canrestimulate the growth of the ECM, and because its fibrin matrixprovides a suitable scaffold for new tissue growth.

Therefore, in accordance with the present invention, there is provided amethod of treating degenerative disc disease in an intervertebral dischaving a nucleus pulposus, comprising:

a) administering autologous uncultured mesenchymal stem cells into thedegenerating disc; and

b) transdiscally administering at least one additional therapeutic agentinto the degenerating disc.

For the purposes of the present invention, “transdiscal administration”includes, but is not limited to:

a) injecting a formulation into the nucleus pulposus of a degeneratingdisc, such as a relatively intact degenerating disc;

b) injecting a formulation into the annulus fibrosus of a degeneratingdisc, such as a relatively intact degenerating disc;

c) providing a formulation in a patch attached to an outer wall of theannulus fibrosus,

d) providing a formulation in a depot at a location outside but closelyadjacent to an outer wall of the annulus fibrosus (“trans-annularadministration”); and

e) providing the formulation in a depot at a location outside butclosely adjacent to an endplate of an adjacent vertebral body(“trans-endplate administration”).

Also in accordance with the present invention, there is provided aformulation for treating degenerative disc disease, comprising:

a) autologous uncultured mesenchymal stem cells; and

b) at least one additional therapeutic agent,

wherein the formulation is present in an amount suitable foradministration into a degenerating disc.

Also in accordance with the present invention, there is provided adevice for delivering a formulation for treating degenerative discdisease to the disc comprising:

a) a chamber containing the formulation comprising autologous unculturedmesenchymal stem cells and at least one additional therapeutic agent;and

b) a delivery port in fluid communication with the chamber and adaptedto administer the formulation to the disc.

In some embodiments, the cells may be introduced (i.e., administered)into the nucleus pulposus or the annulus fibrosus depending on whichextra-cellular matrix needs rebuilding. In other embodiments, the cellsmay be introduced into both regions of the disc. Specific therapeuticagents may be selected depending on the region of the disc where thecells are going to be delivered.

In some embodiments, the cells alone are administered (e.g., injected)into the disc through a needle, such as a small bore needle.Alternatively, the formulation can also be injected into the disc usingthe same small bore needle. In some embodiments, the needle has a boreof about 22 gauge or less, so that the possibilities of producing aherniation are mitigated. For example, the needle can have a bore ofabout 24 gauge or less, so that the possibilities of producing aherniation are even further mitigated.

If the volume of the direct injection of the cells or formulation issufficiently high so as to cause a concern of overpressurizing thenucleus pulposus, then it is preferred that at least a portion of thenucleus pulposus be removed prior to administration (i.e., directinjection) of the mesenchymal stem cells. In some embodiments, thevolume of removed nucleus pulposus is substantially similar to thevolume of the formulation to be injected. For example, the volume ofremoved nucleus pulposus can be within about 80-120% of the volume ofthe formulation to be injected. In addition, this procedure has theadded benefit of at least partially removing some degenerated disc fromthe patient.

When injecting the mesenchymal stem cells into the nucleus pulposus, itis desirable that the volume of drug (i.e., formulation of cellssuspended in growth medium or a carrier) delivered be between about 0.5ml and about 3.0 ml comprising cells suspended in growth medium or acarrier. When injected in these smaller quantities, it is believed thatthe added or replaced volume will not cause an appreciable pressureincrease in the nucleus pulposus. Factors to consider when determiningthe volume of drug to be delivered include the size of the disc, theamount of disc removed and the concentration of the mesenchymal stemcells in the growth medium or carrier.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of treating degenerative disc disease in an intervertebraldisc having a nucleus pulposus and annulus fibrosus in a patient,comprising administering GDF-5 and concentrated autologous unculturedmesenchymal stem cells from 7-10 ml of the patient's bone marrow into adegenerated intervertebral disc immediately following harvesting of thecells.
 2. The method of claim 1, wherein the autologous uncultured cellsare administered to said intervertebral disc using a carrier selectedfrom the group consisting of beads, microspheres, nanospheres,hydrogels, polymers, ceramics, collagen gel and platelet gel.
 3. Themethod of claim 2, wherein the carrier comprises a hydrogel.
 4. Themethod of claim 2, wherein the carrier comprises microspheres.
 5. Themethod of claim 1, wherein said autologous uncultured mesenchymal stemcells are administered into said intervertebral disc in a formulationwith a volume of between about 0.5 ml and about 3.0 ml.
 6. The method ofclaim 1, wherein said autologous uncultured cells are administered intothe nucleus pulposus of the intervertebral disc.
 7. The method of claim1, wherein said autologous uncultured cells are administered into theannulus fibrosus of the intervertebral disc.
 8. The method of claim 1,wherein a portion of the nucleus pulposus is removed prior totransdiscally administering said autologous uncultured cells into thedegenerated intervertebral disc.
 9. The method of claim 1, wherein thecells are administered through a needle.
 10. The method of claim 1,wherein said formulation is administered in an amount of less than 1.0ml.
 11. The method of claim 1, wherein the cells are concentratedyielding an average of 3.6×10⁶ cells per ml in final cell suspensionprior to being administered into the intervertebral disc.
 12. The methodof claim 1, wherein the cells are concentrated by centrifugation. 13.The method of claim 1, wherein the cells are concentrated by filtration.14. The method of claim 1, wherein the GDF-5 and the cells areadministered into the intervertebral disc using a carrier, wherein thecarrier is selected from the group consisting of beads, microspheres,nanospheres, hydrogels, gels, polymers, ceramics, collagen and plateletgels.
 15. The method of claim 1, wherein the GDF-5 is administeredsimultaneously with administering the cells to the disc.
 16. The methodof claim 1, wherein the GDF-5 is administered prior to administering thecells to the disc.
 17. The method of claim 16, wherein the GDF-5 isadministered after administering the cells to the disc.
 18. The methodof claim 1, wherein the GDF-5 is administered after administering thecells to the disc.
 19. A method of treating degenerative disc disease inan intervertebral disc having a nucleus pulposus and annulus fibrosus ina patient, comprising transdiscally administering GDF-5 and autologousuncultured mesenchymal stem cells from the patient's bone marrow into adegenerated intervertebral disc immediately following harvesting of theautologous uncultured mesenchymal stem cells in a formulation with afinal volume of between about 0.5 ml and about 3.0 ml.
 20. The method ofclaim 19, wherein the cells are concentrated yielding an average of3.6×10⁶ cells per mL in final cell suspension prior to beingadministered into the intervertebral disc.
 21. The method of claim 20,wherein the cells are concentrated by centrifugation.
 22. The method ofclaim 20, wherein the cells are concentrated by filtration.
 23. Themethod of claim 19, wherein the cells are administered to the disc usinga carrier, wherein the carrier is selected from the group consisting ofbeads, microspheres, nanospheres, hydrogels, gels, polymers, ceramics,collagen and platelet gels.
 24. The method of claim 19, wherein theGDF-5 and the cells are administered into the intervertebral disc usinga carrier, wherein the carrier is selected from the group consisting ofbeads, microspheres, nanospheres, hydrogels, gels, polymers, ceramics,collagen and platelet gels.
 25. The method of claim 24, wherein thecarrier comprises a hydrogel.
 26. The method of claim 24 wherein thecarrier comprises microspheres.
 27. The method of claim 19, wherein theGDF-5 is administered simultaneously with administering the cells to thedisc.
 28. The method of claim 19, wherein the GDF-5 is administeredprior to administering the cells to the disc.
 29. The method of claim19, wherein the cells are administered into the nucleus pulposus of thedisc.
 30. The method of claim 19, wherein the cells are administeredinto the annulus fibrosus of the disc.
 31. The method of claim 19,wherein a portion of the nucleus pulposus is removed prior toadministering the cells into the intervertebral disc.
 32. The method ofclaim 19, wherein the cells are administered through a needle.
 33. Themethod of claim 32, wherein the needle bore has a maximum gauge of about24 gauge.
 34. The method of claim 19, wherein the formulation isadministered in an amount of less than 1 mL.
 35. The method of claim 19,wherein the cells are provided intra-operatively to the patientfollowing harvesting from the patient.